2-Methyl-3-butenyl-1-pyrophosphoric acid salt and agent for treating lymphocytes

ABSTRACT

A pharmaceutically acceptable salt of 2-methyl-3-butenyl-l-pyrophosphoric acid; an agent for treating lymphocytes which comprises at least one of 2-methyl-3-butenyl-l-pyrophosphoric acid, a pharmaceutically acceptable salt thereof, and a hydrate thereof; Vγ2Vδ2 type T cells treated by the same; and a medicine containing the same specifically stimulate and proliferate the human Vγ2Vδ2 type T cells, and also induce and enhance an antitumor activity thereof.

TECHNICAL FIELD

[0001] The present invention relates to a novel salt of an organicpyrophosphoric acid compound. It also relates to an agent for treatinglymphocytes containing the organic pyrophosphoric acid compound or thesalt thereof which effectively induces and potentiates an antitumoreffect, Vγ2Vδ2 type T cells treated by the agent for treatinglymphocytes and to a medicine containing the same.

BACKGROUND ART

[0002] As a method for inducing and enhancing an antitumor effect inhuman lymphocytes, it has been known LAK therapy using interleukin-2.That is, it has been known that by having about 800 U/ml ofinterleukin-2 to act on the lymphocytes, cell groups induced therebywith an antitumor activity can be used as an antitumor effecter.However, in this method, there are serious side effects, for example,destruction of the self cells such as endovascular cells due tonon-specific cytotoxic property possessed by LAK cells and the like orinduction of autoimmunity due to non-specific activation of T cells byinterleukin-2, thereby making it difficult to apply this methods to anactual clinical field.

[0003] As compounds which specifically activate Vγ2Vδ2 type T cells,there have been known mycobacteria-derived isopentenyl pyrophosphoricacid and mono ethyl phosphoric acid obtained by an organic synthesis. Inthose methods, however, the concentrations of those compounds arerequired to be several hundreds of μM to several mM in order to activateVγ2Vδ2 type T cells. Such high concentrations of the compounds may havea toxic effect on the cells, therefore, it was difficult to use thosecompounds to induce and enhance an antitumor effect of the lymphocytesin a large scale. In any case, it has not yet been known a syntheticcompound which can act on the Vγ2Vδ2 type T cells in a concentration ofseveral hundreds of nM to several hundreds of μM, and specificallyproliferate those cell groups.

[0004] The present invention has been aimed to solve the above-mentionedproblems of the prior art. An object of the present invention is toprovide a novel compound that can specifically stimulate and proliferatethe human Vγ2Vδ2 type T cells, an agent for treating lymphocytes thatinduces and/or potentiates an antitumor effect of the human Vγ2Vδ2 typeT cells, Vγ2Vδ2 type T cells treated by the same, and a medicine whichcomprises the same.

SUMMARY OF THE INVENTION

[0005] The present inventors have made extensive and intensive studiesin search for a compound that can specifically stimulate and proliferatethe human Vγ2Vδ2 type T cells thereby inducing and enhancing theantitumor effect thereof. As a result, they have found apharmaceutically acceptable salt of 2-methyl-3-butenyl-1-pyrophosphoricacid, especially a sodium salt thereof as a novel organic pyrophosphoricacid compound. That is, they have found that, when lymphocytes in humanblood such as peripheral blood or the lymph are treated by such anorganic pyrophosphoric acid compound, Vγ2Vδ2 type T cells arespecifically stimulated and proliferated whereby the antitumor effectthereof are induced and potentiated, and thus, the present invention hasbeen completed.

[0006] Accordingly, the present invention relates to a pharmaceuticallyacceptable salt of 2-methyl-3-butenyl-1-pyrophosphoric acid which is anovel organic pyrophosphoric acid compound, especially a sodium saltthereof and to an agent for treating lymphocytes which comprises atleast one selected from the group consisting of2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof, especially a sodium salt thereof, and a hydrate thereof.Further, the present invention relates to Vγ2Vδ2 type T cells treated bythe agent for treating lymphocytes, and a medicine which comprises thesame.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 is a chromatogram showing an elution curve of the sodiumsalt of 2-methyl-3-butenyl-1-pyrophosphoric acid synthesized in Exampleby Q sepharose HP column chromatography using 820 nm absorption as aparameter.

[0008]FIG. 2 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when Vγ2Vδ2 type T cells treated in Test 1acted on Daudi cells.

[0009]FIG. 3 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when Vγ2Vδ2 type T cells treated in Testexample 2 acted on EJ-1 cells.

[0010]FIG. 4 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when Vγ2Vδ2 type T cells treated in Testexample 3 acted on T24 cells.

[0011]FIG. 5 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when Vγ2Vδ2 type T cells treated in Testexample 4, subsequently frozen for preservation and thawed, acted onDaudi cells.

[0012]FIG. 6 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when Vγ2Vδ2 type T cells treated in Testexample 5, subsequently frozen for preservation and thawed, acted onEJ-1 cells.

[0013]FIG. 7 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when Vγ2Vδ2 type T cells treated in Testexample 6, subsequently frozen for preservation and thawed, acted on T24 cells.

[0014]FIG. 8 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when each of the Vγ2Vδ2 type T cells collectedfrom the peripheral blood of A, treated, frozen for preservation andthawed and Vγ2Vδ2 type T cells collected from the peripheral blood of Fand treated respectively acted on Daudi cells.

[0015]FIG. 9 is a graph showing a relation of a specific lysis ratiorelative to an E/T ratio when each of the Vγ2Vδ2 type T cells collectedfrom the peripheral blood of A, treated, frozen for preservation andthawed and Vγ2Vδ2 type T cells collected from the peripheral blood of Fand treated respectively acted on normal peripheral blood cells of A.

BEST MODE FOR CARRYING OUT THE INVENTION

[0016] The novel pyrophosphoric acid compound of the present inventionis a pharmaceutically acceptable salt of2-methyl-3-butenyl-1-pyrophosphoric acid, and sodium salt is arepresentative. The sodium salt is represented by the formula (I):

[0017] (wherein each of X independently represents a hydrogen atom or asodium atom, at least one of which is a sodium atom). It is preferablethat 2 of X are sodium atoms due to the compatibility to in vivo cells.Further, it can contain water of crystallization. As a pharmaceuticallyacceptable salt, there may be mentioned a potassium salt, an ammoniumsalt, a triethyl ammonium salt and an amino acid salt such as lysinesalt in addition to the above-described sodium salt.

[0018] 2-Methyl-3-butenyl-1-pyrophosphoric acid sodium salt of thepresent invention can be synthesized, for example, in a method describedas follows. That is, 2-methyl-3-butenyl-1-ol is reacted in a solventsuch as acetonitrile with bis (triethyl ammonium) phosphoric acid usingtrichloroacetonitrile as a catalyst. The obtained reaction product isseparated with diethyl ether and an aqueous ammonia solution, then, theproduct extracted in the diethyl ether layer is applied to an anionexchange column chromatography, followed by elution from triethylammonium bicarbonate buffer by a concentration gradient method, toobtain 2-methyl-3-butenyl-1-pyrophosphoric acid. Subsequently, it istreated with a Na type cation exchange resin to obtain a sodium salt.Other pharmaceutically acceptable salts can be synthesized according tothe above method.

[0019] The thus obtained sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid is white powder withdeliquenscence, which becomes a white viscous substance when it containswater. When it is dissolved in a neutral aqueous solution, for example,at pH 6 to 7, it can be stably preserved more than one week at roomtemperature, and more than one year at −20° C. However, in an acidicrange of pH 4 or less, dissociation of inorganic pyrophosphoric acid isobserved within a few minutes.

[0020] 2-Methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceuticallyacceptable salt thereof, especially a sodium salt thereof and a hydratethereof specifically stimulate and proliferate the human Vγ2Vδ2 type Tcells existing in the human blood such as the peripheral blood or thehuman lymph, and at the same time, they promote induction andenhancement of antitumor activity of those cells. As thepharmaceutically acceptable salt, the above-mentioned salts areexemplified.

[0021] Accordingly, when the human blood or lymph is treated with anagent for treating lymphocytes of the present invention containing atleast one kind of the above mentioned organic pyrophosphoric acidderivatives, it specifically stimulates and proliferates the humanVγ2Vδ2 type T cells, and at the same time, it can induce and enhance theantitumor activity thereof. In this case, an analysis using the humanVγ2Vδ2 type T cell clones shows that 2-methyl-3-butenyl-1-organicpyrophosphoric acid compound, especially the above described novelsodium salt can stimulate the cells even in a very small amount such asseveral hundreds nM to several tens μM, and proliferate the human Vγ2Vδ2type T cells in the presence of a very small amount of several hundredsμM, thereby inducing and enhancing the antitumor activity thereof.

[0022] As an organic pyrophosphoric acid compound to be contained as anactive component in the agent for treating lymphocytes of the presentinvention, it is exemplified by the above-mentioned2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof and a hydrate thereof. Among those, a sodium salt ispreferable due to a good compatibility to in vivo cells, and a sodiumsalt wherein 2 of X are sodium atoms in the above-mentioned formula (I)is especially preferable. Those salts can be used in combination withouta need for isolation, in a proper pH range to meet the requirements,preferably in a range of pH 6 to 7

[0023] When the agent for treating lymphocytes containing theabove-mentioned organic pyrophosphoric acid compound is acted on thehuman blood, especially on the peripheral blood, if interleukin-2 isadded as a cofactor in a concentration of 1 to 20 U/ml, specificproliferation of the Vγ2Vδ2 type T cells becomes outstanding. As long asthe concentration of the interleukin-2 is limited under 20 U/ml,non-specific activation of the lymphocytes would not occur differentfrom the case of the LAK cells. Further, it is possible to use othercofactor such as interleukin-15. The human Vγ2Vδ2 type T cells have akind of natural killer activity. The present invention is completelydifferent from conventional induction methods of the antitumor activityin that it can proliferate those cell groups antigen-specifically.

[0024] That is, in the LAK therapy and the TIL therapy, the cellproliferation factor such as intrerleukin-2 is added excessively therebyforcibly activating naive cell groups in the peripheral blood.Therefore, it is accompanied by side effects such as attacking the selfcells. On the contrary, the above-mentioned organic pyrophosphoric acidcompound is naturally to be used to proliferate the population of theVγ2Vδ2 type T cells having natural killer activity, therefore, it is notexcessively and forcibly enhancing the antitumor activity by using anexcess amount of the antigen. The concentration of 20 μM of theabove-mentioned organic pyrophosphoric acid type antigen corresponds tothe activity of 10 to 50 U, and is appropriate as a concentration.Therefore, proliferation and activation of the human Vγ2Vδ2 type T cellsand induction and enhancement of the antitumor activity thereof by thepresent invention is immunochemically appropriate causing no destructionof the self cells. From the above, the agent for treating lymphocyte ofthe present invention is a more advantageous agent for inducing andenhancing the antitumor activity due to its higher specificity and lessside effects in comparison to the LAK therapy and the TIL therapy.

[0025] In addition, the use of the agent for treating lymphocytes of thepresent invention is advantageous in that it is possible to freeze thetreated cells. That is, the blood containing lymphocytes and/or thelymph are collected at the suitable point and preserved by freezing thenas necessity arises, the lymphocytes are activated by theabove-mentioned organic pyrophosphoric acid compound. By proliferatingthe Vγ2Vδ2 type T cells as mentioned above, and by inducing andenhancing the antitumor activity thereof, it is possible to decrease aburden for donors of peripheral blood lymphocytes.

[0026] Further, since the human Vγ2Vδ2 type T cells have natural killeractivity, even if it is preserved by freezing after the antitumoractivity is induced and potentiated, it can exhibit the antitumoractivity immediately after it thaws. This makes it possible to use thecell at any time. On the contrary, the LAK therapy and the TIL therapyrequire fresh cells all the time, and every time the antitumor activityis induced and potentiated, the lymphocytes should be collected and theproliferation factor should be added. From the above-mentioned facts,the therapy using the agent for treating lymphocytes of the presentinvention is clearly more advantageous than the LAK therapy and the TILtherapy. Therefore, the present invention enables the wide range ofantitumor therapy in the practical medical field by using a medicinewhich comprises the human Vγ2Vδ2 type T cells whose antitumor activityis induced and potentiated.

[0027] The Vγ2Vδ2 type T cells which are treated and proliferated withthe agent for treating lymphocytes of the present invention, and whoseantitumor activity is induced and potentiated can exhibit the antitumoractivity by returning those T cells themselves into the human patient asthe peripheral blood. The administration method includes a localinjection, intravenous injection, endermic absorption and the like.

[0028] The thus treated Vγ2Vδ2 type T cells are free from MHC limitationand it is possible to administer them to the other person.

[0029] Therefore, the present invention includes the Vγ2Vδ2 type T cellswhich are treated with the agent for treating lymphocytes of the presentinvention and a medicine containing the same.

[0030] When the compound of the present invention or the agent fortreating lymphocytes containing the same is administered as a medicine,it can be prepared by a general preparation technique, and it can beused as a solid or liquid preparation form such as a tablet, a capsule,powders, granules, a suppository, cream, an ointment, an aqueoussolution, an emulsion, oil or a suspension and the like.

[0031] Further, in that case, except for preparations whose pH is 4 orless, generally used additives in the preparation can be used asnecessity arises, such as an excipient, a disintegrator, a lubricant, abinder, a preservative, a stabilizer, an osmotic pressure adjuster, asubstrate and the like.

[0032] The examples of the above-mentioned additives include glucose,lactose, starch, carboxymethyl cellulose, magnesium stearate, talc,liquid paraffin, polyvinyl alcohol, vegetable oil, polyalkylene glycol,and the like. It can also contain a medical component in addition tothese.

EXAMPLES

[0033] The present invention will be explained in more detail referringto Examples and Test examples hereinbelow. These examples, however, arenot construed to limit the scope of the present invention.

[0034] In Test examples, treatment of the peripheral blood by2-methyl-3-butenyl-1-pyrophosphoric acid or by a salt thereof wasconducted as follows unless otherwise specified. Lymphocytes containedin the peripheral blood derived from healthy volunteer were treated sothat the final concentration of the pyrophosphoric acid compound was 20μM, and cultured in a Yssel's medium under conditions of temperature at37° C. and a carbon dioxide concentration of 5%. After 2, 4, 6, 8 and 10days, interleukin-2 was added in an amount of 10 U/ml, respectively, asa cofactor. After 12 days, the cells were collected.

Example

[0035] (Synthesis of Sodium Salt of 2-methyl-3-butenyl-1-pyrophosphoricacid)

[0036] To 1 mol of 2-methyl-3-butenyl-1-ol was added 1 mol oftrichloroacetonitrile as a catalyst, and while stirring at a temperatureof 25° C., 4 mol of bis (triethyl ammonium) phosphoric acid dissolved in20 ml of acetonitrile was added dropwise over 4 hours and reacted whilestirring for another 2 hours. To the reaction product was added 100 mlof diethyl ether and the mixture was transferred to a separation funnel.100 ml of 0.88% aqueous ammonia was added thereto and shaken, and theobtained organic pyrophosphoric acid was extracted in an organic layer.After diethyl ether was evaporated from the organic layer under areduced pressure, the resultant liquid was subjected to Q Sepharose HPanion exchange column chromatography having a diameter of 2.5 cm and alength of 8 cm, then washed with water.

[0037] Subsequently, using a triethyl ammonium bicarbonate buffer, theorganic pyrophosphoric acid compound was eluted by a concentrationgradient method from 0 to 500 mM. An elution curve of the columnchromatography with absorption at 820 nm as a parameter is shown inFIG. 1. Here, a fraction eluted in an area of approximately 150 to 200mM was 2-methyl-3-butenyl-1-pyrophosphoric acid. After freeze-dried thisfraction, it was dissolved in 2 ml of water and the solution was treatedby Na type Dowex 50 W cation exchange resin (Trade name of Dow ChemicalCo.) to obtain a sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidcontaining a little amount of water as a white viscous substance. It wasdried to obtain a sodium salt of 2-methyl-3-butenyl-1-pyrophosphoricacid as white powder.

Test Example 1

[0038] Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by the treatment of the human peripheral bloodderived from healthy volunteers A, B and C were conducted according tothe above-mentioned method. The peripheral blood containing thepotentiated human Vγ2Vδ2 type T cells were acted on Daudi cells toanalyze cytotoxicity. The results are shown in FIG. 2. In FIG. 2, thetransverse axis shows an effecter/target ratio (hereinafter refer to asan E/T ratio), that is, a ratio of existing human Vγ2Vδ2 type T cellsand Daudi cells. The vertical axis shows a specific lysis ratio.

[0039] As is apparent from FIG. 2, in both case of the peripheral bloodof A and B, cytotoxicity reached a plateau even when the E/T ratio is1:1, exhibiting a high cytotoxic effect. In the case of the peripheralblood of C, cytotoxicity reached the same level when the E/T ratio is5:1.

Test Example 2

[0040] Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by treatment of the human peripheral bloodderived from healthy volunteers A, B and C were conducted in the samemanner as in Test example 1 according to the above-mentioned method. Theperipheral blood containing the potentiated human Vγ2Vδ2 type T cellswere acted on EJ-1 cells to analyze cytotoxicity. The results are shownin FIG. 3. In FIG. 3, the transverse axis shows an E/T ratio, and thevertical axis shows a specific lysis ratio.

[0041] As is apparent from FIG. 3, in both cases of the peripheral bloodof A and B, cytotoxicity reached a plateau when the E/T ratio is 5:1,and in case of the peripheral blood of C, cytotoxicity reachedapproximately a plateau when the E/T ratio is 10:1.

Test Example 3

[0042] Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by treatment of the human peripheral bloodwere conducted according to the above-mentioned method. The peripheralblood containing the potentiated human Vγ2Vδ2 type T cells were acted onT24 cells to analyze cytotoxicity. The results are shown in FIG. 4. InFIG. 4, the transverse axis shows an E/T ratio, and the vertical axisshows a specific lysis ratio

[0043] As is apparent from FIG. 4, in all cases of the peripheral bloodsof A, B and C, cytotoxicity reached approximately a plateau when the E/Tratio is 20:1.

Test Example 4

[0044] Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by treatment of the human peripheral bloodwere conducted according to the above-mentioned method using theperipheral blood of the volunteer A and other healthy volunteers D and EThus obtained treated peripheral blood was preserved by freezing for oneyear at −20° C. The treated and preserved blood after being thawed wereacted on Daudi cells to analyze cytotoxicity. The results are shown inFIG. 5. In FIG. 5, the transverse axis shows an E/T ratio, and thevertical axis shows a specific lysis ratio.

[0045] As is shown by FIG. 5, in both cases of the peripheral bloods ofA and E, cytotoxicity reached approximately a plateau when the E/T ratiois 20:1, and in a case of the frozen-preserved peripheral blood of D,cytotoxicity reached a plateau when the E/T ratio is 20:1 while there isa difference in degree. As a result, it is evident that the treatedlymphocytes maintain sufficient antitumor effect even after they arepreserved by freezing.

Test Example 5

[0046] Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells bytreatment of the human peripheral blood and induction and enhancement aswell as preservation by freezing and thawing were conducted in the samemanner as in Test example 4. The treated and preserved blood was actedon the EJ-1 cells to analyze cytotoxicity. The results are shown in FIG.6. In FIG. 6, the transverse axis shows an E/T ratio, and the verticalaxis shows a specific lysis ratio.

[0047] As is apparent from FIG. 6, cytotoxicity reached approximately aplateau when the E/T ratio is 20:1 in any case while there is adifference in degree. And it is observed that the antitumor effect wasmaintained even after the cells are preserved by freezing.

Test Example 6

[0048] Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells bytreatment of the human peripheral blood and induction and enhancement aswell as preservation by freezing and thawing were conducted in the samemanner as in Test example 4. The treated and preserved blood was actedon the T24 cells to analyze cytotoxicity. The results are shown in FIG.7. In FIG. 7, the transverse axis shows an E/T ratio, and the verticalaxis shows a specific lysis ratio.

[0049] As is apparent from FIG. 7, cytotoxicity reached approximately aplateau when the E/T ratio is 20:1 in any case while there is adifference in degree. And it is observed that the antitumor effect wasmaintained even after the cells were preserved by freezing.

Test Example 7

[0050] Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Test example and using the peripheral blood of the volunteerA, proliferation of the human Vγ2Vδ2 type T cells by treatment of thehuman peripheral blood and induction and enhancement were conductedaccording to the above-mentioned method. In the same manner as in Testexample 4, the treated and preserved blood which had been preserved byfreezing and thawed were acted on Daudi cells to analyze cytotoxicity.On the other hand, peripheral blood of other healthy volunteer F wastreated according to the above-mentioned method and they were acted onDaudi cells in the same manner to analyze cytotoxicity. The results areshown in FIG. 8. Further, using the treated and preserved blood derivedfrom A that had been preserved by freezing and thawed and the treatedperipheral blood derived from F, cytotoxicity was analyzed based on thenormal peripheral blood cells of A. The results are shown in FIG. 9. Inthose Figs., the transverse axis shows an E/T ratio, and the verticalaxis shows a specific lysis ratio.

[0051] As is apparent from FIG. 8, cytotoxicity reached approximately aplateau when E/T ratio is 20:1 in both cases of the peripheral bloods ofA (after preservation by freezing and thawing) and F.

[0052] On the other hand, from the results for the peripheral bloods ofA (after preservation by freezing and thawing) and F in FIG. 9, it isevident that the Vγ2Vδ2 type T cells whose antitumor activity wasinduced and potentiated by the sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid do not attack the normal cellswhen they are used as they are or even after they are preserved byfreezing and thawed.

Test Example 8

[0053] 10 ml of the peripheral blood collected from a healthy volunteerG was subjected to Ficoll-Paque specific gravity centrifugation topurify the peripheral blood mononuclear cells and they were suspended ina Yssel's medium. The cells were apportioned into a 24-hole plate sothat the number of the cells becomes a concentration of 2.5 million/1.5ml/hole, and then, according to the above-mentioned method, treatments 1to 5 were conducted as shown in Table 1. Among them, treatment 1 is atreatment using only a culture medium and treatment 2 solely usesinterleukin-2, and both treatments are for comparison. Treatments 3 to 5are treatments of the present invention using the sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid obtained in Example andinterleukin-2 in combination. On the day 11 after conducting thetreatment, a ratio of Vγ2Vδ2 type T cells in existing CD3 cells wascalculated. The results are shown in Table 1. TABLE 1 Treatment Sodiumsalt of 2- Vγ2Vδ2 type methyl-3-butenyl- T cells/ Treatment1-pyrophosphoric Interleukin-2 CD3 cells No. acid [μM] [U/ml] [%]  1* —— 6.7  2* — 10 6.9 3 2 10 58.3 4 20 10 85.5 5 200 10 94.5

[0054] As is apparent from Table 1, when interleukin-2 was used solely,the ratio of Vγ2Vδ2 type T cells was approximately the same level asthat of the treatment using only the culture medium. On the contrary, inthe treatments 3 to 5 of the present invention, proliferation of theVγ2Vδ2 type T cells by the sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid was significant and the ratioswere increased accompanying an increase of the amount of thepyrophosphoric acid compound added.

[0055] Industrial Applicability

[0056] As is explained above, 2-methyl-1-butenylpyrophosphoric acid, apharmaceutically acceptable salt thereof, especially the sodium salt of2-methyl-3-butenylpyrophosphoric acid of the present invention and/or anagent for treating lymphocytes containing a hydrate thereof as an activecomponent stimulate and proliferate the human Vγ2Vδ2 type T cells, andat the same time, exhibit a specific effect to induce and enhance theantitumor effect thereof. In addition, they do not exhibit anycytotoxicity on the self cells. They can specifically activate a naturalkiller cell such as the human Vγ2Vδ2 type T cell with a low antigenconcentration.

[0057] The agent for treating lymphocytes of the present invention canbe applied to various clinical fields. For example, peripheral blood ofa cancer patient or a healthy person is collected and treated by theagent for treating lymphocytes of the present invention to stimulateVγ2Vδ2 type T cells, thereby proliferating them polyclonally as well asinducing and enhancing the antitumor activity thereof. By re-circulatingthose lymphocytes in the body, the antitumor activity in vivo can beexpected. In that case, it is possible to preserve the lymphocytes byfreezing, they can be frozen and preserved at a suitable stage, and canbe administered to the patient as a need arises.

What is claimed:
 1. Vγ2Vδ2 type T cells treated by an agent for treatinglymphocytes comprising an agent for treating lymphocytes containing atleast one of 2-methyl-3-butenyl-1-pyrophosphoric acid, apharmaceutically acceptable salt thereof or a hydrate thereof.
 2. TheVγ2Vδ2 type T cells of claim 1, wherein the2-methyl-3-butenyl-1-pyrophosphoric acid salt is a sodium salt.
 3. TheVγ2Vδ2 type T cells of claim 1, wherein the agent further comprisesinterleukin-2.
 4. The Vγ2Vδ2 type T cells of claim 2, wherein the agentfurther comprises interleukin-2.
 5. A pharmaceutical compositioncomprising Vγ2Vδ2 type T cells treated with an agent for treatinglymphocytes containing at least one of2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof or a hydrate thereof and a pharmaceutically acceptablecarrier.
 6. The pharmaceutical composition of claim 5, wherein thecomposition further comprises interleukin-2.
 7. A method of treating apatient in need thereof comprising administering an effective amount ofa pharmaceutical composition comprising Vγ2Vδ2 type T cells treated withan agent for treating lymphocytes containing at least one of2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof or a hydrate thereof and a pharmaceutically acceptablecarrier.
 8. A method of treating human lymphocytes, comprising: treatingperipheral blood from a human with an agent which comprises at least oneof 2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceuticallyacceptable salt thereof and a hydrate thereof, thereby stimulatingVγ2Vδ2 type T cells present in said perpheral blood.
 9. The method ofclaim 7, which further comprises the step of polyclonally proliferatingcells in said blood.